i a lysine- and histidine-specific amino acid transporter ... · plant physiol. (1 997) 11 5: 27-1...

Plant Physiol. (1 997) 11 5: 11 27-1 134

LHTl I A Lysine- and Histidine-Specific Transporter in Arabidopsis

Amino Acid

Lishan Chen’ and Daniel R. Bush*

Program in Physiological and Molecular Plant Biology, University of lllinois (L.C., D.R.B.); Photosynthesis Research Unit, United States Department of Agriculture-Agricultura1 Research Service (D.R.B.); and Department

of Plant Biology, University of Illinois, 196 Madigan Laboratory, 1201 West Gregory Drive, Urbana, lllinois 61 801 (D.R.B.)

We have identified a new amino acid transporter from the Ara- bidopsis fhaliana expressed sequence tag cDNA collection by functional complementation o f a yeast amino acid transport mutant. Transport analysis of the expressed protein i n yeast shows that it is a high-affinity transporter for both lysine (Lys) and histidine with Michaelis constant values of 175 and 400 p ~ , respectively. This transporter (LHTl, lysine histidine transporter) has l itt le affinity for arginine when measured directly i n uptake experiments or indi- rectly with substrate competition. The cDNA i s 1.7 kb with an open reading frame that codes for a protein with 446 amino acids and a calculated molecular mass of 50.5 kD. Hydropathy analysis shows that LHTl i s an integral membrane protein with 9 to 10 putative membrane-spanning domains. Southern-blot analysis suggests that LHT7 is a single-copy gene in the Arabidopsis genome. RNA gel-blot analysis shows that this transporter i s present in all tissues, with the strongest expression in young leaves, flowers, and diques. Whole- mount, in situ hybridization revealed that expression i s further localized on the surface of roots in young seedlings and in pollen. Overall, LHTl belongs to a new class of amino acid transporter that is specific for Lys and histidine, and, given i t s substrate specificity, it has significant promise as a tool for improving the Lys content of Lys-deficient grains.

~~ ~~

Long-distance transport of resources and information is a fundamental process in a11 multicellular organisms. In higher plants sugars and amino acids are transported from the sites of primary assimilation to heterotrophic tissues via mass flow in the phloem. The import-dependent tissue systems, which include roots, young leaves, developing fruits, and specialized storage organs, carry out many es- sentia1 processes required for plant growth (Pate, 1980; Gifford et al., 1984; Thorne, 1985; Schubert, 1986). Long- distance transport of organic nutrients is also an essential activity during seed germination and grain filling (Thorne, 1985; Fisher and Macnicol, 1986), in symbiotic N, assimilation (Schubert, 1986; Mylona et al., 1995), and in nutrient recycling during senescence (Thomas and Stoddart, 1980; Feller and Fischer, 1994). In a11 cases integral membrane proteins that mediate sugar and amino acid transport

Present address: Department of Microbiology, University of

* Corresponding author; e-mail dbush&iuc.edu; fax 1-217-244- Washington, Seattle, WA 98195.

4419.

across the plasma membrane are essential components of the resource-allocation system in higher plants.

Amino acids are the predominant form of nitrogen avail- able to the heterotrophic tissues of the plant. As such, they are the precursors of many essential molecules, including proteins, nucleic acids, chlorophyll, phytohormones, phy- toalexins, phenylpropanoids, accessory pigments, and lig- nin. Amino acids are actively transported into plant cells by proton-coupled symporters (Bush, 1993). Bush and Langston-Unkefer (1988) used isolated membrane vesicles and imposed proton electrochemical potentials to provide the first biochemical description of proton-coupled amino acid transport in plants. Subsequently, Li and Bush (1990, 1991) provided a detailed analysis of the transport properties and bioenergetics of severa1 amino acid symporters using highly purified plasma membrane vesicles isolated from sugar beet leaves. They showed that these transporters are electrogenic, that they are inhibited by DEPC, and that there are at least four classes of symporters based on their preferences for groups of amino acids that share charge or structural similarities. Williams et al. (1992, 1996) and Weston et al. (1995) found similar properties of transporter activity in membrane vesicles isolated from castor bean cotyledons and roots.

Frommer et al. (1993) and Hsu et al. (1993) cloned the first plant amino acid transporter cDNA (NAT2 lAAP1) by functional complementation of yeast amino acid transport mutants. Functional complementation in yeast has become a powerful tool for plant transport biologists because it circumvents the daunting task of purifying these extremely low-abundance membrane proteins using traditional biochemical strategies (Bush, 1993; Frommer et al., 1994; Frommer and Ninneman, 1995; Tanner and Caspari, 1996). Frommer’s group went on to identify five additional transporter cDNAs that are closely related to NAT2 IAAP1, pro- viding good evidence that they represent a family of trans- locator genes (Kwart et al., 1993; Fischer et al., 1995; Rentsch et al., 1996). In addition to the NAT2IAAP1 gene family, a cationic amino acid transporter cDNA, AAT1, that is related to an animal amino acid transporter has been described (Frommer et al., 1995), and an aromatic amino

Abbreviations: DEPC; diethyl pyrocarbonate; EST, expressed sequence tag; Ura, uracil.

1127 www.plantphysiol.orgon September 26, 2020 - Published by Downloaded from Copyright © 1997 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

1128 Chen and

acid transporter (Chen, 1997) and two Pro transporters have also been cloned (Rentsch et al., 1996).

Given the complexity of amino acid transport activities associated with resource allocation during a11 phases of plant growth, we have continued to look for new amino acid transporter genes that encode additional participants of this fundamental physiological process. One approach we have used to achieve this goal has been to survey the Arabidopsis thaliana EST cDNA database (Newman et al., 1994) for sequences that exhibit some similarity to NAT2 / AAPl using a BLAST search protocol (Altschul et al., 1990). Although severa1 of the ESTs identified using this strategy included previously described clones, others represented novel genes that have only small regions of sequence similarity (Chen, 1997). We requested the novel EST cDNAs from the stock center, ligated them into yeast expression vectors, and scored them for amino acid transport activity by testing their ability to complement yeast amino acid transport mutants. In the results reported here we describe a cDNA initially identified in our EST survey that encodes a new class of amino acid transporter found in Arabidopsis.

MATERIALS AND METHODS

Molecular Cloning

EST cDNAs were obtained from the Arabidopsis thaliana EST cDNA library (Ohio State University Arabidopsis Stock Center, Columbus). Inserts were analyzed by restriction digestion and subcloned into the expression vector AYES (Elledge et al., 1991) for complementation of a yeast amino acid transport mutant. AYES contains the URA3 gene as a selectable marker for positive transformants. The EST cDNAs were double-digested with Xbal and EcoRI and then they were directionally cloned into AYES. Standard procedures for ligation and transformation into Escherichia coli followed the methods of Sambrook et al. (1989) or Ausubel et al. (1994).

Yeast Transformation and Complementation

The His auxotroph and transport-deficient mutant of Saccharomyces cerevisiae used in this study (JT16; MATa hip 1-624 his4401 ura3-52 ino2 can2) (Tanaka and Fink, 1985) was a gift from G. Fink (Whitehead Institute, Massachu- setts Institute of Technology, Cambridge). JT16 was main- tained on S1 medium supplemented with 0.2% Ura and without sorbitol (Hsu et al., 1993). Yeast were transformed by electroporation and / or lithium acetate as described by Becker and Guarente (1991) or Gietz et al. (1992), respectively. Complementation of His transport was scored as previously described (Hsu et al., 1993). Urat transformants were selected with S1 medium containing 1 M sorbitol (Hsu et al., 1993). For transport complementation, positive transformants were then plated on a His-limiting medium containing 4% Gal, 0.17% yeast-nitrogen base without amino acids and ammonium salts, 0.5% ammonium sulfate, 0.002% inosine, 0.1% Arg, and 130 FM His. In a11 cases, insert-free AYES and NAT2/ AYES were run in parallel as negative and positive controls, respectively.

Bush Plant Physiol. Vol. 11 5, 1997

Transport Measurements

Yeast transport activity was measured as described by Hsu et al. (1993). Cells were grown to the midlogarithmic phase and then collected by centrifugation. They were resuspended at 200 to 300 mg cells mL-l in transport buffer that contained His-limiting medium without His and Arg (the pH was adjusted to 5.0 with KOH). Ten microliters of cells was used in a 500-pL transport reaction that contained 0.2 to 1.0 pCi of 14C-labeled and unlabeled amino acid to the desired final concentration. At predeter- mined times, 200 pL was removed from the reaction buffer and the cells were collected and washed on a micropore filter. JT16 cells transformed with insert-free AYES were used to measure background transport activity in a11 transport experiments, and results are reported as net transport (i.e. LHT1-expressing cells accumulation minus accumulation by insert-free vector controls).

Molecular Techniques

Yeast colony PCR was performed according to the method of Schneider et al. (1995), and plasmid DNA ex- traction from yeast was adapted from the method of Ward (1990). Two milliliters of saturated yeast culture was centrifuged and cells were resuspended in 200 pL of 2.5 M

LiCI, 50 mM Tris HCl, pH 8.0,4% Triton X-100, and 62.5 mM EDTA, and then 200 pL of phenol-chloroform (l:l, w/v) and 2.0 g of acid-washed glass beads (0.5 mm) were added. This mixture was immediately vortexed in a mini- beadbeater (Biospec, Bartlesville, OK) for 2 min and then centrifuged at 14,000 rpm for 5 min. The upper layer was collected and 600 pL of ethanol was added to precipitate the DNA. Yeast RNA was isolated using a kit according to the manufacturer's instructions (Qiagen, Chatsworth, CA). Dideoxynucleotide sequencing (Sanger et al., 1977) was performed on double-strand DNA templates using Seque- nase version 2.0 according to the manufacturer's instructions (Amersham). Plant DNA was isolated according to the method of Dellaporta et al. (1983). Plant RNA was isolated using guanidinium as described by Kiedrowski et al. (1992). Whole-mount, in situ hybridization of Arabidop- sis seedlings and flowers was as described by Bennett (1994).

For Southern-blot hybridizations plant genomic DNA was digested with different restriction enzymes, separated on a 0.8% agarose gel in TAE buffer (40 mM Tris-acetate and 1 mM EDTA), and depurinated with a 10-min treat- ment in 0.25 M HC1. DNA was transferred onto Hybond N+ membranes (Amersham) in 0.4 M NaOH, and then cross- linked to the membrane using a UV cross-linker (Strat- agene). c~-~'P-Labeled probes were synthesized using ran- dom hexamers (Boehringer Mannheim) or the Megaprimed system (Amersham). Probes were purified with either a Bio-Spin column (Bio-Rad) or a nucleotide-removal kit (Qiaquick, Qiagen). [c~-~~P]Cytosine was purchased from Amersham. The hybridization and wash conditions followed the suggestions of the membrane manufacturer (Amersham). Low-stringency washes consisted of two 10- min washes with 2X SSPE (Sambrook et al., 1989) and 0.1%

www.plantphysiol.orgon September 26, 2020 - Published by Downloaded from Copyright © 1997 American Society of Plant Biologists. All rights reserved.


Lys and His Transporter 1129

SDS at room temperature. The high-stringency wash in-cluded an additional wash with IX SSPE and 0.1% SDS at65°C for 15 min, and two washes of 0.1 x SSPE and 0.1%SDS at 65°C for 30 min.

Twenty micrograms of total RNA was separated on aformaldehyde gel (Zielinski, 1987) and transferred ontonylon membranes (Nytran, Schleicher & Schuell) for RNAgel-blot analysis. a-32P-Labeled probe was prepared as forthe Southern-blot hybridization method. The hybridizationand wash conditions followed the membrane manufactur-er's instruction (Schleicher & Schuell).

RESULTS

An EST cDNA Complements a Yeast Amino AcidTransport Mutant

One approach we have taken to identify additionalamino acid transporters has been to survey the EST cDNAdatabase for clones that exhibit low levels of deducedamino acid sequence similarity to NAT2. Several ESTcDNAs were obtained from the collection at the Ohio StateUniversity Arabidopsis Stock Center because each pos-sessed modest levels of similarity with the amino terminusof NAT2. Each EST cDNA that was sufficiently long topotentially contain a complete open reading frame wassubcloned into a yeast expression vector (Elledge et al.,1991) and then transformed into a yeast strain auxotrophicfor His and in which the high-affinity His transporter wasdeleted. Transformants that acquired the expression vectorwere selected on a high-His medium without Ura (Fig. 1 A).The vector allows for growth in the absence of Ura becauseit contains the URA3 gene as a selectable marker.

Ura+ transformants were subsequently scored for theirability to complement the His-transport deficiency of theyeast mutant by growing them on a low-His medium thatdoes not support growth of the deletion mutant (Hsu et al.,1993). Of several EST cDNAs tested, only LHT1 allowed forgrowth under His-limiting conditions (Fig. IB). Althoughthis result suggests that LHT1 encodes a protein with His-transport activity, there are alternative explanations forthis observation. For example, a suppressor mutation in theyeast genome could allow for His uptake or the EST cDNAcould encode a homolog to the mutated His biosynthesisgene (H/S4). We tested for suppressor mutants by substi-tuting Glc for Gal in the selection medium. Because Glcrepresses the GALl promotor engineered into the expres-sion vector, shifting the C source to Glc eliminated expres-sion of the plant protein. LHT1 -containing transformantstransferred to the Glc-based medium lost their ability tocomplement His-limited growth, suggesting that comple-mentation is attributable to the encoded plant proteinrather than to a suppressor mutation (Fig. 1C). The LHT1transformants also failed to grow on a His-free Gal me-dium, suggesting that the expressed plant protein is not aH/S4 homolog in the biosynthetic pathway (Fig. ID). Thesedata suggest that LHT1 is a plant amino acid transporter.

Figure 1. Complementation of JT16 by LHT1/AYES. The LHT1 cDNAwas subcloned into AYES and transformed into JT16 to test forfunctional complementation of the transport-deficient phenotype.Transformants were selected on Ura-free plates in the presence ofhigh His (A). Ura+ cells were transferred to low-His plates to test theirability to complement the His-transport mutation (B). Dependence ofthe suppressed phenotype on the expressed plant protein was dem-onstrated by testing transformants on Glc plates. This C sourcerepresses the GALl promotor of the expression vector (C). Verifica-tion that complementation was the result of newly acquired transportactivity versus His biosynthesis was confirmed by transferring cells toa His-free medium (D). NAT2 (a previously cloned amino acidtransporter) and insert-free AYES were run in parallel as positive andnegative controls, respectively (data not shown).

LHT1 Is a Lys- and His-Specific Transporter

Amino acid transport activity of LHT1 was investigatedusing 13 different amino acids as potential substrates. Lysand His transport were the most active among all of thesubstrates examined (Fig. 2). Although LHT1 exhibitedsome transport capacity for a few other amino acids, suchas Leu, it was most effective at transporting Lys and His,hence the name Lys and His Transporter 1, LHT1.

Lys and His transport by LHT1 displayed saturable,concentration-dependent uptake kinetics that were consis-tent with carrier-mediated transport, and the double-reciprocal plots of those data gave Km values of 175 and 400JU.M, respectively (Fig. 3). The apparent Km for Leu was 11mM (data not shown), which is higher than typical physi-ological ranges. The substrate specificity of the transporterwas further demonstrated when Arg (at a 10-fold excess)did not decrease Lys transport in a substrate competitionexperiment (Fig. 4). Lys transport was sensitive to carbonylcyanide m-chlorophenylhydrazone, which is consistentwith transport coupled to the proton motive force (Fig. 4).Transport was also inhibited by DEPC, although an indi-rect effect on the yeast cell cannot be ruled out (Fig. 4). www.plantphysiol.orgon September 26, 2020 - Published by Downloaded from

Copyright © 1997 American Society of Plant Biologists. All rights reserved.


1130 Chen and Bush Plant Physiol. Vol. 11 5, 1997

2, Amino Acid Transported

Figure 2. Amino acid transport in LHTl -expressing cells. Thirteen amino acids were tested as potential substrates for LHTl . Each amino acid was tested at 100 p ~ . Transport activity in cells containing insert-free vector was also determined for each amino acid and results are reported as LHTl -dependent transport (i.e. LHTl- expressing cell accumulation minus insert-free cell accumulation). Lys transport was 0.39 pmol mg-’ fresh weight min-’.

Earlier biochemical descriptions of amino acid transport into purified plasma membrane vesicles implicated a DEPC-sensitive His residue(s) in the reaction mechanism (Li and Bush, 1992).

LHTl Belongs to a New Class of Transport Protein

The cDNA insert of positive transformants was 1664 bp long. It contained an open reading frame encoding a protein of 446 amino acids with a calculated molecular mass of

40 - - c ‘2 30- 3

: 20-

E

cc z F 10-

O

a

3

I I I I I I

-5 O 5 10 15 20 25

1/s (PM> Figure 3. Double-reciproca1 plot of Lys and His transport kinetics by LHTl -expressing cells. Transport activity in cells containing insert- free vector was also determined for each amino acid, and results are reported as LHTl -dependent transport (i.e. LHTl -expressing cell accumulation minus insert-free cell accumulation). The apparent K,,, for Lys was 175 p~ and for His it was 400 pM.

2

1.5

3 ru

\

O

E 0.5

O O 2 4 6

Time (min)

Figure 4. Effect of Arg and chemical inhibition on Lys transport by LHTl . Lys transport (O) was not affected by a 1 O-fold excess concentration of Arg (O). DEPC (O) and carbonyl cyanide m- chlorophenylhydrazone (.) inhibited Lys uptake. Transport solutions included 100 p~ Lys in the presence or absence of I mM Arg, 2 mM DEPC, or 1 O p~ carbonyl cyanide m-chlorophenylhydrazone.

50.5 kD (Fig. 5A). Hydropathy analysis of the deduced sequence (Kyte and Doolittle, 1982) suggests that LHTl is a hydrophobic integral membrane protein, with 9 to 10 putative transmembrane domains (Fig. 5B). The proposed topology of LHTl is different from that predicted for other plant amino acid transporters, suggesting that it represents a new class of carrier. The NATZIAAP family, the Pro transporters, and a cationic amino acid transporter a11 have 10 or more putative transmembrane domains (Frommer et al., 1993, 1995; Hsu et al., 1993; Kwart et al., 1993; Fischer et al., 1995; Rentsch et al., 1996). A phylogenetic comparison of LHTl with amino acid transporters from a variety of organisms also suggests that LHTl is not part of a previously described family of transporters (Fig. 6). It should be noted, however, that LHTl is closely related to a putative amino acid transporter gene isolated from tobacco (Lalanne et al., 1995). Our analysis suggests that LHTl represents a new class of plant amino acid transporters, although it does share some similarity with amino acid transporters identified in Caenovhabditis elegans, yeast, and plants.

LHT7 1s a Single-Copy Gene in the Arabidopsis Cenome and 1s Preferentially Expressed on the Root Surface and in Siliques

Southern-blot hybridization suggests that LHTZ is a single-copy gene in the Arabidopsis genome (Fig. 7). RNA gel-blot analysis showed that LHTZ is transcribed into a 1.7-kb message. Moreover, the pattern of tissue-specific expression suggests that LHTZ is preferentially expressed in flowers, young leaves, and siliques, although it is also present in older leaves, stems, and roots (Fig. 8). Whole- mount, in situ analysis showed that expression is further localized to t h e surface of the root in young seedlings and in anthers (data not shown).




1 MVAQAEHDCH31 TSSKNAHAWY61 MSQLOGPGI91 HEMVPGKRFD121 PQQLIVEIGV151 DDCKPIKLTY181 ISGSFSCCCR211 RRSIRLQSEN241 GHNWLEIQA271 AYIWALCYF301 SLKKPAWLIA331 VFEMMETLLV361 AATMFVGMTF391 FLPCVIWIAI421 LFLMVLSPIG

QECEKLAAAR QKEIEDWLPI 30SAFHNVTAMV GAGVLGLPVA 60AVLVLSWVIT LYTDWrjlVEM 90RYHELGQHAF GEKD3LYIW 120CIWMVTOGK SLKKFHELVC 150FDHFASVHF VLSHLPNFNS 180YVSQLLttlRM GIISKQRCSR 210NSRWFNFFS GLGCWAFAYA 240TIPSTPEKPS KGBWRGVTV 270PVALVGYYIF OCVEOnlM 300TMJIFWIHV IGSVQBSMP 330KKLNFRPTIT LRFFVRNFW 360PFFQGLIAFF GOFAFAPTTy 390YKPKKYSLSW WANWVCIVFG 420GLRTIVIQAK GYKFYS 446

Figure 5. Deduced amino acid sequence andhydropathy plot of LHTl. The LHTl cDNA en-codes a protein containing 446 amino acids (A).The Kyte and Doolittle (1982) hydropathy anal-ysis shows that LHT1 is a hydrophobic mem-brane protein with 9 to 10 putative transmem-brane helices (B).

B

200 300

100 200 300Amino acid position from N to C terminal

400

400

DISCUSSION

LHTl represents a new class of amino acid transportersin plants because it has limited similarity to previouslydescribed transporters and because its predicted mem-brane topology differs from that of the other transporters.The similarity of LHTl to the NAT2/AAP gene family isstrongest around the first transmembrane domain, and thatis why this EST cDNA showed up in our initial BLASTsearch of the EST database (Altschul et al., 1990). We haveobserved that this is a highly conserved domain shared bymany eukaryotic amino acid transporters (Hoffmann, 1985;Ahmad and Bussey, 1986; Frommer et al., 1993; Hsu et al..

Figure 6. Phylogeny comparison of LHTl with related amino acidtransporters. LYP1 is the Lys-specific transporter in yeast (Sychrovaand Chevallier, 1993); CAN1 is the cationic amino acid transporterin yeast (Hoffmann, 1985; Ahmad and Bussey, 1986); LysP is theLys-specific permease in E. coli (Steffes et al.,1992); NSAAP1 is aputative amino acid transporter in tobacco (Lalanne et al., 1995);NAT2/AAP1 is a neutral amino acid transporter in Arabidopsis (From-mer et al., 1993; Hsu et al., 1993); Hurr is the human cationic aminoacid transporter (Yoshimoto et al., 1991; Christensen, 1992); andAAT1 is an Arabidopsis cationic amino acid transporter (Frommer etal., 1995). This phylogeny was generated with MegAlign version 1.05(DNASTAR, Madison, Wl) using the Clustal method with a PAM250residue weight table and it is rooted assuming a biological clock.

1993; Fischer et al., 1995; Rentsch et al., 1996) and anArabidopsis aromatic amino acid transporter (Chen, 1997).We believe that future investigations will uncover the func-tional significance of this region.

LHTl is easily differentiated from a functionally relatedcationic amino acid transporter, AAT1, which was recentlydescribed in Arabidopsis (Frommer et al., 1995). AAT1 has

Length 1 2 3 4(Kb)

I12.2—8.1—

5.0—

3.0—

2.0—1.6—

1.0—

0.5—1

Figure 7. Southern-blot analysis of LHTL Arabidopsis genomic DNAwas digested with BamHI (lane 2), fcoRI (lane 3), and H/ndlll (lane4). Lane 1 was a 1 -kb ladder. The LHTl cDNA has an internal H/ndlllsite. The results shown are for a high-stringency wash. Low-stringency washes gave a similar pattern. www.plantphysiol.orgon September 26, 2020 - Published by Downloaded from



1132 Chen and Bush Plant Physiol. Vol. 115, 1997

LHTl

ActinFigure 8. Expression pattern of LHTl. RNA gel-blot analysis of LHTlgave a single band at 1.75 kb. The actin gene was used as a loadingcontrol. RNA was isolated from siliques (lane 1), flowers (lane 2), oldand young leaves (lanes 3 and 4, respectively), stems (lane 5), androots (lane 6). The intensity of the LHTl signal was weak relative toactin (2-d exposure versus 2 h).

533 amino acid residues, which is much larger than LHTl(446 amino acids). In addition, AAT1 contains 14 putativetransmembrane domains (Frommer et al., 1995), whereasour analysis suggests that LHTl is composed of 9 to 10putative transmembrane helices (Fig. 5B). This is also lessthan the 11 transmembrane domains we have mapped inthe NAT2/AAP1 gene family (Chang and Bush, 1997). Webelieve that LHTl belongs to a unique class of amino acidtransporters in Arabidopsis. We realize, however, thatmore detailed analysis of the evolutionary relationshipsamong the plant amino acid transporters may uncover acommon ancestral progenitor. A putative amino acid trans-porter recently cloned in tobacco appears to be a LHTlortholog (Lalanne et al., 1995).

Two basic amino acid transporters, CAN1 (Hoffmann,1985; Ahmad and Bussey, 1986) and LYP1 (Sychrova andChevallier, 1993), have been identified in budding yeast.CAN1 encodes a peptide containing 590 amino acids andLYP1 encodes one with 611 amino acids. Although bothwere predicted to contain 12 transmembrane domains,gene-fusion experiments with CAN1 suggest that it con-tains only 10 membrane-spanning regions (Ahmad andBussey, 1988). The Lys transporter (LysP) in E. coli is alsolonger than LHTl (489 residues; Steffes et al., 1992), andgene-fusion experiments have identified 12 transmem-brane domains (Ellis et al., 1995). LHTl is more closelyrelated to the yeast and E. coli transporters than to AAT1(Fig. 6).

An intriguing feature of the basic amino acid transport-ers we have examined is that the amino-terminal domainsare highly acidic regions that contain many Glu and Aspresidues. The acidic amino acid content for the aminoterminus of these porters is 25% for LHTl, 19% for LYP1,24% for CAN1, and 15% for LysP. Steffes et al. (1992)proposed that a glutamate residue, E-16, in LysP may playa role in substrate binding. LHTl contains a glutamateresidue at the E-25 position that may correspond to theE-16 position in LysP. Mutations in this position of thetransporter will help define the role of these acidic residuesin basic amino acid transporters. In addition to this acidicregion, LHTl also shares two conserved His's found in theNAT2/AAP gene family. These His residues have beenmutated and shown to be essential for NAT2/AAP1 func-tion (Chen, 1997).

Many of the plant amino acid transporters identified sofar have wide substrate specificity, although they often

favor a subgroup of related amino acids with lower Kmvalues and higher rates of transport. LHTl is somewhatintermediate in this characteristic in that it is a particularlyactive Lys and His transporter. The other amino acidstested had less than 35% of the Lys transport activity, andeven the Km for the third most-active substrate, Leu, was25-fold higher than that for His (11 mM, data not shown).Although Leu clearly moves through LHTl, we do notthink it is a major substrate for this transporter, becauseplant cells generally contain low levels (si mM) of thisamino acid (Winter et al., 1992).

The substrate specificity of LHTl distinguishes it fromAAT1, the functionally related Arabidopsis cationic aminoacid transporter, and from basic amino acid transport ac-tivities previously described in barley, castor bean, andtobacco (Soldal and Nissen, 1978; Berry et al., 1981; Har-rington and Henke, 1981; Bright et al., 1983; Weston et al.,1995). More recently, two transport mutants were de-scribed in Arabidopsis (rltll and raecl) that have reducedLys transport in the substrate concentration range in whichLHTl is active. Thus, it is possible that one of them couldbe a LHTl mutant (Heremans et al., 1997). AAT1 transportsboth Lys and Arg, as did the amino acid transport activitiesdescribed in barley and tobacco. In contrast, LHTl is not aneffective Arg transporter. This was demonstrated by thelow rates of transport over a range of pH values (pH4.0-7.0; Fig. 2 and data not shown). In addition, Arg wasnot a competitive inhibitor of Lys transport by LHTl, evenwhen it was present at 10 times the concentration of Lys(Fig. 4). AAT1 substrate competition analysis showed thatArg was an excellent competitor for Lys transport in thattransporter (Frommer et al., 1995). Although LHTl andAAT1 are both basic amino acid transporters in Arabidop-sis, their transport properties clearly differentiate them asunique amino acid transporters. This observation supportsour earlier investigations with purified membrane vesicles,which suggested the presence of two cationic amino acidtransport systems based on biphasic transport kinetics (Liand Bush, 1990).

Both RNA gel-blot analysis and whole-mount, in situhybridization indicate that LHTl is most strongly ex-pressed in pollen, siliques, and on the root surface (Fig. 8).We infer from this expression pattern that LHTl may beinvolved in Lys transport into heterotrophic tissue systems.This conclusion is supported by the observation that therelated tobacco ortholog was isolated from a pollen-specificlibrary (Lalanne et al., 1995). The functionally related cat-ionic amino acid transporter (AATI) identified in Arabi-dopsis is most strongly expressed in vascular tissue andflowers (Frommer et al., 1995). The expression patterns ofLHTl and AAT1 complement one another, suggesting thatLHTl may be involved in nutrient uptake in sink tissues,whereas AAT1 may contribute to long-distance transport.

LHTl is an excellent candidate for using biotechnologyto modify the nutritional value of harvested tissues (Bush,1998). For example, many cereals are poor sources of pro-tein for humans and animals because they are low in Lys(Kriz and Larkins, 1991; Galili, 1995). One strategy formodifying the nutritional value of these deficient crops www.plantphysiol.orgon September 26, 2020 - Published by Downloaded from




would be to use targeted expression of LHTl to enhance the Lys content of harvested seed. Targeted expression of LHTl in the phloem of mature leaf tissue may increase the amount of Lys and His transported per unit of C. This would increase the Lys and His content of developing seeds when the Lys-enriched translocate is released from the phloem. The success of this approach is dependent on severa1 unknown variables. For example, reciproca1 in- creases in mesophyll synthesis and release must occur as these amino acids are actively transported into the phloem, and sink tissues must be capable of storing the excess free amino acids. There is a precedent for increased rates of synthesis under increased demand; Heldt’s work (Riens et al., 1991; Winter et al., 1992) suggests that the mesophyll amino acid pools are dynamically linked to phloem loading and, thus, active loading by a high-affinity transporter may shift the equation to increased synthesis. Likewise, many cells store excess amino acids in the vacuole. We are cur- rently generating transgenic plants to test this approach.

Received April 21, 1997; accepted July 25, 1997. Copyright Clearance Center: 0032-0889/97/115/ 1127/08. The accession number for the sequence reported in this article is

U39782.

LITERATURE ClTED

Ahmad M, Bussey H (1986) Yeast arginine permease: nucleotide sequence of the C A N l gene. Curr Genet 10: 587-592

Ahmad M, Bussey H (1988) Topology of membrane insertion in vitro and plasma membrane assembly in vivo of the yeast arginine permease. MOI Microbiol 2: 627-635

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J MOI Biol 215: 403410

Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1994) Current Protocols in Molecular Biol- ogy. Greene and John Wiley & Sons, New York

Becker DM, Guarente L (1991) High-efficiency transformation of yeast by electroporation. Methods Enzymol 194: 182-187

Bennett MJ (1994) Whole mount in situ method. Weeds World 1:

Berry SL, Harrington HM, Bernstein RL, Henke RR (1981) Amino acid transport into cultured tobacco cells. Planta 153: 511-518

Bright SWJ, Kueh JSH, Rognes SE (1983) Lysine transport in two barley mutants with altered uptake of basic amino acids in the root. Plant Physiol 7 2 821-824

Bush DR (1998) Proton-coupIed sugar and amino acid transporters in plants. Annu Rev Plant Physiol Plant Mo1 Biol44: 513-542

Bush DR (1987) Amino acid transport. In BK Singh, ed, Plant Amino Acids: Biochemistry and Biotechnology. Marcel Dekker, New York (in press)

Bush DR, Langston-Unkefer PJ (1988) Amino acid transport into membrane vesicles isolated from zucchini. Plant Physiol 88: 487490

Chang H-C, Bush DR (1997) Topology of NAT2: a prototypical example of a new family of amino acid transporter. J Biol Chem 272: (in press)

Chen L (1997) Amino acid transporters in Arabidopsis thaliana. PhD thesis. University of Illinois at Urbana-Champaign, Urbana

Christensen HN (1992) A retrovirus uses a cationic amino acid transporter as a cell surface receptor. Nutr Rev 50: 4748

Dellaporta ST, Wood J, Hicks JB (1983) A plant DNA miniprepa- ration: version 11. Plant MOI Biol Reprod 4: 19-21

Elledge SJ, Mulligan JT, Ramer SW, Spottswood M, Davis RW (1991) AYES: a multifunctional cDNA expression vector for the isolation of genes by complementation of yeast and Escherichia coli mutations. Proc Natl Acad Sci USA 88: 1731-1735

12-13

Ellis J, Carlin A, Steffes C, Wu J, Liu J, Rosen BP (1995) Topo- logical analysis of the lysine-specific permease of Escherichiu coli. Microbiology 141: 1927-1935

Feller U, Fischer A (1994) Nitrogen metabolism in senescing leaves. Crit Rev Plant Sci 13: 241-273

Fischer WN, Kwart M, Hummel S, Frommer WB (1995) Substrate specificity and expression profile of amino acid transporters (AAPs) in Arabidopsis. J Biol Chem 270: 16315-16320

Fisher DB, Macnicol PK (1986) Amino acid composition along the transport pathway during grain filling in wheat. Plant Physiol

Frommer WB, Hummel S , Riesmeier JW (1993) Expression cloning in yeast of a cDNA encoding a broad specificity amino acid permease from Arabidopsis thaliana. Proc Natl Acad Sci USA 90:

Frommer WB, Hummel S, Unseld M, Ninneman O (1995) Seed and vascular expression of a high affinity transporter for cationic amino acids in Arabidopsis. Proc Natl Acad Sci USA 9 2

Frommer WB, Kwart M, Hirner B, Fischer WN, Hummel S, Ninneman O (1994) Transporters of nitrogenous compounds in plants. Plant MOI Biol 26: 1651-1670

Frommer WB, Ninneman O (1995) Heterologous expression of genes in bacterial, fungal, animal and plant cells. Annu Rev Plant Physiol Plant Mo1 Biol 46: 419444

Galili G (1995) Regulation of lysine and threonine synthesis. Plant Cell 7: 899-906

Gietz D, St Jean A, Woods RA, Schiestl RH (1992) Improved methods for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20: 1425

Gifford RM, Thorne JH, Hitz WD, Giaquinta RT (1984) Crop productivity and photoassimilate partitioning. Science 225:

Harrington HM, Henke RR (1981) Amino acid transport into cultured tobacco cells. I. Lysine transport. Plant Physiol 67:

Heremans B, Borstlap AC, Jacobs M (1997) The r l t l l and raecl mutants of Arabidopsis thaliana lack the activity of a basic-aminoacid transporter. Planta 201: 219-226

Hoffmann W (1985) Molecular characterization of the C A N l locus in Saccharomyces cerevisiae. J Biol Chem 21: 11831-11837

Hsu LC, Chiou TJ, Chen L, Bush DR (1993) Cloning a plant amino acid transporter by functional complementation of a yeast amino acid transport mutant. Proc Natl Acad Sci USA 90: 7441-7445

Kiedrowski S, Kawalleck P, Hahlbrock K (1992) Rapid activation of a nove1 plant defense gene is strictly dependent on the Ara- bidopsis vpml disease resistant locus. EMBO J 13: 46774684

Kriz AL, Larkins BA (1991) Biotechnology of seed crops: genetic engineering of seed storage proteins. HortScience 26: 1036-1041

Kwart M, Hirner B, Hummel S, Frommer WB (1993) Differential expression of two related amino-acid transporters with differing substrate specificity in Arabidopsis thaliana. Plant J 4: 993-1002

Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mo1 Biol 157: 105-132

Lalanne E, Vede1 F, de Paepe R (1995) Isolation of a Nicotiana sylvestris cDNA (accession no. U31932) encoding an amino acid transporter homologous to Arabidopsis thaliana amino acid permeases (PGR95-069). Plant Physiol 109: 722

Li Z-C, Bush DR (1990) ApH-dependent amino acid transport into plasma membrane vesicles isolated from sugar beet (Beta vul- garis L.) leaves. I. Evidence for carrier-mediated, electrogenic flux through multiple transport systems. Plant Physiol 94: 268-277

Li Z-C, Bush DR (1991) ApH-dependent amino acid transport into plasma membrane vesicles isolated from sugar beet (Beta vul- garis L.) leaves. 11. Evidence for multiple aliphatic, neutra1 amino acid symporters. Plant Physiol 96: 1338-1344

Li Z-C, Bush DR (1992) Structural determinants in substrate rec- ognition by proton-amino acid symports in plasma membrane vesicles isolated from sugar beet leaves. Arch Biochem Biophys

8 2 1019-1023

5944-5948

12036-12040

801-808

373-378

294: 519-526



1134 Chen and Bush Plant Physiol. Vol. 11 5, 1997

Mylona P, Pawlowski K, Bisseling T (1995) Symbiotic nitrogen fixation. Plant Cell 7: 869-885

Newman T, de Bruijn FJ, Green P, Keegstra K, Kende H, McIn- tosh L, Ohlrogge J, Raikhel N, Somerville S, Thomashow M, and others (1994) Genes galore: a summary of methods for accessing results from large-scale partia1 sequencing of anony- mous Arabidopsis cDNA clones. Plant Physiol 106: 1241-1255

Pate JS (1980) Transport and partitioning of nitrogenous solutes. Annu Rev Plant Physiol 31: 313-340

Rentsch D, Hirner B, Schmelzer E, Frommer WB (1996) Salt stress-induced proline transporters and salt stress-repressed broad specificity amino acid permeases identified by suppres- sion of a yeast amino acid permease-targeting mutant. Plant Cell

Riens B, Lohaus G, Heineke D, Heldt HW (1991) Amino acid and sucrose content determined in the cytosolic, chloroplastic, and vacuolar compartments and in the phloem sap of spinach leaves. Plant Physiol 97: 227-233

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463- 5467

Schneider BL, Seufert W, Steiner B, Yang QH, Futcher AB (1995) Use of polymerase chain reaction epitope tagging for protein tagging in Saccharomyces cerevisiae. Yeast 11: 1265-1274

Schubert KR (1986) Product of biological nitrogen fixation in higher plants: synthesis, transport, and metabolism. Annu Rev Plant Physiol37: 539-574

Solda1 T, Nissen P (1978) Multiphasic uptake of amino acids by barley roots. Physiol Plant 43: 181-188

Steffes C, Ellis J, Wu J, Rosen BP (1992) The LysP gene encodes the lysine-specific permease. J Bacteriol 174: 3242-3249

8: 1437-1446

Sychrova H, Chevallier MR (1993) Cloning and sequencing of the Saccharomyces cerevisiae gene LYPl coding for a lysine-specific permease. Yeast 9: 771-782

Tanaka J, Fink GR (1985) The histidine permease gene (HIPI) of Saccharomyces cerevisiae. Gene 38: 205-214

Tanner W, Caspari T (1996) Membrane transport carriers. Annu Rev Plant Physiol Plant Mo1 Biol 47: 595-626

Thomas H, Stoddart JL (1980) Leaf senescence. Annu Rev Plant Physiol 31: 83-111

Thorne JH (1985) Phloem unioading of C and N assimilates in developing seeds. Annu Rev Plant Physiol 36: 317-343

Ward AC (1990) Single-step purification of shuttle vectors from yeast for high frequency back-transformation into E . cozi. Nu- cleic Acids Res 18: 5319

Weston K, Hall JL, Williams LE (1995) Characterization of aminoacid transport in Xicinus communis roots using isolated membrane vesicles. Planta 196: 166-173

Williams LE, Bick JA, Neelam A, Weston KN, Hall JL (1996) Biochemical and molecular characterization of sucrose and amino acid carriers in Ricinus communis. J Exp Bot 47: 1211- 1216

Williams LE, Nelson SJ, Hall JL (1992) Characterization of solute/ proton cotransport in plasma membrane vesicles from Ricinus cotyledons, and a comparison with other tissues. Planta 186:

Winter H, Lohaus G, Heldt HW (1992) Phloem transport of amino acids in relation to their cytosolic levels in barley leaves. Plant Physiol 99: 996-1004

Yoshimoto T, Yoshimoto E, Meruelo D (1991) Molecular cloning and characterization of a nove1 human gene homologous to the murine ecotropic retroviral receptor. Virology 185: 10-17

Zielinski RE (1987) Calmodulin mRNA in barley. Apparent regulation by cell proliferation and light. Plant Physiol 84: 937-943

541-550



i a lysine- and histidine-specific amino acid transporter ... · plant physiol. (1 997) 11 5: 27-1...

Documents